Mechanical ventilation (MV) is used clinically to sustain pulmonary gas exchange in patients that are incapable of maintaining adequate alveolar ventilation. Although MV is often a life- saving intervention, prolonged MV gives rise to problems in weaning patients from the ventilator. Although several factors can contribute to difficult weaning, weak inspiratory muscles (i.e., diaphragm) are a major factor. In this regard, MV results in diaphragmatic inactivity which promotes the rapid development of diaphragmatic atrophy and contractile dysfunction which is known as ventilator- induced diaphragm dysfunction (VIDD). At present, the mechanism(s) responsible for VIDD are poorly understood and thus, no clinical therapy exists. We recently discovered that endurance exercise training results in diaphragmatic adaptations and a diaphragm phenotype that is protected against VIDD. The cellular mechanism(s) responsible for this exercise-induced protection against VIDD remain unknown and are the focus of this application. HYPOTHESIS: Guided by our preliminary experiments, we will test the hypothesis that exercise- induced protection of the diaphragm against VIDD is dependent on increased diaphragmatic levels of: 1) mitochondrial antioxidants (i.e., manganese superoxide dismutase (SOD2) and glutathione) and/or 2) heat shock protein 72 (HSP72). APPROACH: Our hypothesis will be tested using an established animal model of MV. Cause and effect will be determined using innovative molecular and pharmacological tools to manipulate SOD2, GSH, and HSP72 within the diaphragm. SPECIFIC AIMS: 1) To determine if exercise-induced protection against VIDD requires increased levels of SOD2 and/or glutathione within mitochondria of diaphragm muscle fibers; and 2) To establish if overexpression of HSP72 in the diaphragm is necessary and sufficient for exercise-induced protection against VIDD. SIGNIFICANCE: Determining the mechanism(s) responsible for exercise-induced protection of the diaphragm will identify novel molecular targets that can be manipulated pharmacologically, leading to new approaches to prevent VIDD and reduce problems in weaning patients from MV. 1